Chinese Academy Of Agricultural Sciences Thesis

Peste des petits ruminant is a highly contagious disease of domestic and wild small ruminants caused by peste des petits ruminants virus(PPRV).The molecular mechanism of PPRV replication and its interactions with hosts are poorly studied.The objectives of this study were to contribute to the understanding of PPRV replication and related virus/host interaction;and to analyze the functional role of PPRV phosphoprotein in the process of PPRV replication and host immune evasion.Using molecular techniques,the global effect of PPRV infection on Vero cell line and the preliminary pathways explored by PPRV to overcome hosts cellular immunity were investigated.Through our results,we demonstrated the ability of PPRV to dephosphorylate eIF2? in Vero cells infected with PPRV at the multiplicity of infection MOI= 3 analyzed for 36,48,60 and 72 h post-infection(h.p.i).Overexpression of PPRV P protein in mammal cells such as HEK293 T cells demonstrated the potentiality of PPRV P protein to induce the host cellular growth arrest DNA damage protein(GADD34)which is known to be associated with eIF2? dephosphorylation.Further,we discovered that PPRV P protein alone could block PERK/eIF2? phosphorylation.Likewise,chemical treatment with a selective inhibitor of eIF2? dephosphorylation(Salubrinal)followed by analysis of protein expression and mRNA level quantitation resulted in impaired PPRV replication.From the above,we speculate that PPRV exploits eIF2? dephosphorylation to facilitate viral replication and PPRV P protein is involved in this molecular mechanism.Next,it is known that in response to viral infection,hosts cells specific signaling pathways called unfolded proteins response(UPR)may be activated or inhibited to counteract viral induced ER stress to maintain proteostasis.In this study,using Western blot and immunofluorescence analysis,it was revealed that PPRV infection could activate the ATF6 arm of the UPR but not IRE1/XBP1.Furthermore,assembly of stress granules(SGs)which are the temporal cytoplasmic aggregates of stalled translation complexes involved in mRNA storage triggered by the phosphorylation of eIF2? is an important antiviral mechanism.Consider that,both PPRV and PPRV P protein could inhibit eIF2? phosphorylation,we were prompted to check for the effect of PPRV on SG formation.It is known that,during SGs assembly,the primarily aggregates occur through several RNAbinding proteins such as Ras-GAP SH3 domain-binding protein(G3BP1)and T-cell intracellular antigen 1(TIA-1)related protein(TIAR).SGs induction can be visualized by staining cells with known SGs markers such as G3BP1 and TIA-1.Investigation of the effect of PPRV infection on SGs assembly was conducted on Vero cells infected at MOI= 3 for 72 h followed by exposure to different stressors(Arsenite and DTT)for 1 h at 72 h.p.i.to induce oxidative and ER stress SGs formation respectively.Our results revealed that,both PPRV infection and PPRV P protein transfection could inhibit SGs formation,supporting that PPRV and PPRV P protein do inhibit eIF2? phosphorylation and the implication of PPRV P protein in host immune counteraction.In overall,this work provides new sights towards further understanding of PPRV pathobiology,virus replication and its viral/host interactions.